Optimizing total RNA extraction method for human and mice samples.

ABSTRACT

Background: Extracting high-quality total RNA is pivotal for advanced RNA molecular studies, such as Next-generation sequencing and expression microarrays where RNA is hybridized. Despite the development of numerous extraction methods in recent decades, like the cetyl-trimethyl ammonium bromide (CTAB) and the traditional TRIzol reagent methods, their complexity and high costs often impede their application in small-scale laboratories. Therefore, a practical and economical method for RNA extraction that maintains high standards of efficiency and quality needs to be provided to optimize RNA extraction from human and mice tissues. Method: This study proposes enhancements to the TRIzol method by incorporating guanidine isothiocyanate (GITC-T method) and sodium dodecyl sulfate (SDS-T method). We evaluated the effectiveness of these modified methods compared to the TRIzol method using a micro-volume UV-visible spectrophotometer, electrophoresis, q-PCR, RNA-Seq, and whole transcriptome sequencing. Result: The micro-volume UV-visible spectrophotometer, electrophoresis, and RNA-Seq demonstrated that the GITC-T method yielded RNA with higher yields, integrity, and purity, while the consistency in RNA quality between the two methods was confirmed. Taking mouse cerebral cortex tissue as a sample, the yield of total RNA extracted by the GITC-T method was 1,959.06 ± 49.68 ng/mg, while the yield of total RNA extracted by the TRIzol method was 1,673.08 ± 86.39 ng/mg. At the same time, the OD260/280 of the total RNA samples extracted by the GITC-T method was 2.03 ± 0.012, and the OD260/230 was 2.17 ± 0.031, while the OD260/280 of the total RNA samples extracted by the TRIzol method was 2.013 ± 0.041 and the OD260/230 was 2.11 ± 0.062. Furthermore, q-PCR indicated that the GITC-T method achieved higher yields, purity, and greater transcript abundance of total RNA from the same types of animal samples than the TRIzol method. Conclusion: The GITC-T method not only yields higher purity and quantity of RNA but also reduces reagent consumption and overall costs, thereby presenting a more feasible option for small-scale laboratory settings.